Program Director/Principal Investigator (Last, First, Middle): Kukar, Thomas L. PROJECT SUMMARY (See instructions): Numerous lines of evidence support the hypothesis that selective targeting of A[unreadable]42 is an ideal therapeutic strategy to prevent and possibly treat Alzheimer's disease (AD), the major cause of dementia among the elderly. A class of compounds called ?-secretase modulators (GSMs) is being pursued as AD therapeutics because they are able to selectively alter A[unreadable]42 levels>. The first GSMs to be discovered were certain non- steroidal anti-inflammatory drugs (NSAIDs), which have been shown to selectively decrease A[unreadable]42 without global inhibition of APR processing. As whole GSMs minimally alter total Abeta production and instead shift the gamma-secretase cleavage site. The mechanism of GSMs has not been conclusively proven and different explanations for their activity have been proposed including: 1) allosteric binding to ?-secretase 2) inhibition of the Rho-ROCK signaling pathway 3) conformational changes in presenilin or 4) decreased dimerization of APR. We have recently discovered, using novel GSM photoaffinity probes, that these drugs do not label the y-secretase enzyme but instead modulate cleavage by binding to a the substrate, APP. We hypothesize that binding of APR by GSMs shifts the} position of APP-CTF in the membrane resulting in altered gamma-secretase cleavage. This hypothesis will be tested through the following specific aims: 1) investigate how substrate targeting by GSMs produces a shift in the cleavage pattern of A[unreadable] using a combination of molecular biology and protein biochemistry 2) determine the specificity of GSMs for affecting APP proteolysis by ?-secretase in comparison to other substrates using mass spectrometry and 3) test if unnatural amino acids can be used to study proteolysis of APP-CTF[unreadable] by ?-secretase. Aim 1 and career/technical training will take place during the mentored phase, which will facilitate the execution of Aims 2 and 3 during the independent phase. These studies should provide additional insight into the mechanisms whereby NSAIDs and other GSMs shift A[unreadable] cleavage and how they exert their protective effects in vivo. This work will also guide future efforts to design more potent GSMs which will be useful as chemical probes for understanding the biology of ?-secretase and as potential therapeutics for Alzheimer's disease. RELEVANCE (See instructions): Alzheimer's disease (AD) is the most common type of dementia afflicting the elderly with no known cure. ?- secretase modulators (GSMs) are a promising class of drugs under investigation to treat AD. The proposed research will provide critical insight into how GSMs work and may ultimately lead to improved AD therapeutics.